Recombinant DNA molecules encoding aminopeptidase enzymes and their use in the preparation of vaccines against helminth infections

• US 6,534,638 B1
• Filed: 08/05/1998
• Issued: 03/18/2003
• Est. Priority Date: 05/08/1992
• Status: Expired due to Fees

• Current Assignee: Barbraham Institute
• Original Assignee: Barbraham Institute
• Inventors: Smith, Trevor Stanley, Knox, David Patrick, Oliver, Joanna Jane, Newton, Susan Elizabeth, Munn, Edward Albert, Graham, Margaret
• Primary Examiner(s): MINNIFIELD, NITA M
• Application Number:
  US09/129,366
• Time in Patent Office: 1,686 Days
• Field of Search: 536/23.1, 536/23.2, 536/23.4, 536/23.7, 435/69.1, 435/69.3, 435/69.7, 435/71.1, 435/71.2, 435/320.1, 435/243
• US Class Current: 536/23.1
• CPC Class Codes: A61K 2039/55566 Emulsions, e.g. Freund's adjuvant, MF59
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/0003 Invertebrate antigens
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: A61P 33/10 Anthelmintics
• CPC Class Codes: C07H 13/04 having the esterifying carboxyl radicals attached to acyclic carbon atoms
• CPC Class Codes: C07K 14/4354 from nematodes
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 9/00 Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
• CPC Class Codes: C07K 9/001 the peptide sequence having less than 12 amino acids and not being part of a ring structure
• CPC Class Codes: C12N 2799/026 where the vector is derived from a baculovirus
• CPC Class Codes: C12N 9/48 acting on peptide bonds (3.4)

Abstract

The present invention provides nucleic acid molecules containing nucleotide sequences encoding helminth aminopeptidase enzymes, and antigenic fragments and functionally-equivalent variants thereof, their use in the preparation of vaccines for use against helminth parasites, and synthetic polypeptides encoded by them.

Claims

1. An isolated nucleic acid comprising at least one nucleotide sequence which encodes a helminth aminopeptidase or an antigenic portion thereof, said nucleic acid selected from the group consisting of i) any sequence of SEQ ID NOs:

1-15 or 9-21, ii) a sequence fully complementary to said sequence of (i), iii) a portion of said sequence of (i) wherein said portion of said sequence encodes an antigenic peptide of said helminth aminopeptidase, and (iv) a sequence which hybridizes with said sequence of (i) or (ii) under conditions of 2×

SSC, 65°

C. (where SSC=0.15 M NaCl, 0.015 M sodium citrate, pH 7.2) and which has at least 60% identity with said sequence of (i).

4. An expression or cloning vector comprising a nucleic acid molecule as defined in claim 1, 2 or 3.

5. An isolated prokaryotic or eukaryotic cell containing a nucleic acid molecule as defined in claim 1, 2 or 3.

2. An isolated nucleic acid comprising at least one nucleotide sequence which encodes a polypeptide which raises protective antibodies against helminth parasites, which sequence incorporates at least one aminopeptidase-encoding sequence, selected from the group consisting of i) any sequence of SEQ ID NOS:

1-15 or 19-21, ii) a sequence fully complementary to said sequence of (i), iii) a portion of said sequence of (i), and iv) a sequence which hybridizes with said sequence of (i) or (ii) under conditions of 2×

SSC, 65°

C. (where SSC=0.15 M NaCl, 0.015 M sodium citrate, pH 7.2) and which has at least 60% identity with said sequence of (i).

6. A method for preparing a synthetic polypeptide comprising an amino acid sequence of an aminopeptidase enzyme wherein the amino acid sequence is encoded by i) at least one of SEQ ID NOS:

1-5 or 19-21, ii) a sequence complementary to said sequence of (i), iii) a portion of said sequence of (i), or iv) a sequence which hybridizes to at least one sequence of (i) or (ii) under conditions of 2×

SSC, 65°

C. (where SSC=0.15 M NaCl, 0.015 M sodium citrate, pH 7.2) and which has at least 60% identity with said sequence, wherein the method comprises culturing a prokaryotic or eukaryotic cell containing a nucleic acid molecule as defined in claim 2 under conditions whereby said synthetic polypeptide is expressed, and recovering said synthetic polypeptide thus produced.

3. An isolated nucleic acid comprising at least one nucleotide sequence which corresponds to or which is complementary to one or more sequences selected from the group of sequences consisting of i) M1(SEQ ID NO:

1), ii) B1A (SEQ ID NO;

2), iii) B1A-3′

(SEQ ID NO;

3), iv) B2 (SEQ ID NO;

4), v) M1AUS (SEQ ID NO;

5), vi) AusB1 (SEQ ID NO;

6), vii) 014-015 (2.5 PCR) (SEQ ID NO;

7), viii) 014-872 (3.5 PCR clone

     2) (SEQ ID NO;

8), ix) A-648 (5′

END of B1) (SEQ ID NO;

9), x) A-650 (5′

-end of 2.5 PCR) (SEQ ID NO;

10), xi) a-649 (5′

end of 3.5 PCR) (SEQ ID NO;

11), xii) 014-178 (3′

end of AustB1 clone

     2) (SEQ ID NO;

12), xiii) 014-178 (3′

end of AustB1 clones 3 and

     6) (SEQ ID NO;

13,) xiv) 014-872 (3.5 PCR clone

     10) (SEQ ID NO;

14), xv) 014-872 (3.5 PCR clone

     19) (SEQ ID NO;

15), xvi) H11-1 (SEQ ID NO;

19), xvii) H11-2 (SEQ ID NO;

20), xviii) H11-3 (SEQ ID NO;

21), and xix) a sequence which hybridizes with any of said sequences of (i)-(xviii) under conditions of 2×

SSC, 65°

C. (where SSC=0.15 M NaCl, 0.015 M sodium citrate, pH 7.2) and which has at least 60% identity with said sequence of (i)-(xviii).